SK7332000A3 - Methods for treating postmenopausal women using ultra-low doses of estrogen - Google Patents

Abstract

The present invention provides methods for treating physical conditions resulting from postmenopausal estrogen decline in a postmenopausal subject, and in particular methods for reducing the risk of osteoporotic bone fractures in a postmenopausal subject. The present invention also provides a kit useful for carrying out the methods of the present invention

Description

Technical field

The invention relates to the use of estrogen for the manufacture of a medicament for the treatment of physical conditions caused by a decrease in estrogen in subjects undergoing transition and kits for administering such medicaments.

BACKGROUND OF THE INVENTION

Endogenous estrogens decrease dramatically after natural or surgical menopause and this decrease causes a significant increase in bone loss and subsequent fractures. Endogenous estrogens are clearly important in maintaining the skeletal health of younger women. However, the importance of endogenous estrogens in older women is less doubtful.

It has previously been shown that endogenous estrone levels in women over 65 were not associated with subsequent hip or vertebral fractures. S.R. Cummings, et al., J. Bone Min. Res. 10 (Suppl I): S174 (1995). Estradiol, however, is a more potent estrogen than estrone, and studies of its relationship to fractures were inconclusive. Serum estradiol levels in women prior to transition are on average above 100 pg / ml. Serum oestradiol levels in women not undergoing hormone replacement therapy fall below 20 pg / ml, and up to 30 to 50 percent of post-transition women have serum estradiol levels that are undetectable by conventional susceptible test methods (i.e., less than 5 pg / ml). Conventional treatment of postmenopausal women includes estrogen replacement therapy at doses sufficient to maintain serum estradiol levels above 40-60 pg / ml.

Conventional hormone replacement therapy has been shown to be useful in the treatment of physical conditions caused by a decrease in estrogen following transition, including a reduction in loss or even an increase in bone density; and decreasing the risk of bone fractures. However, studies have suggested that hormone replacement therapy may be associated with an increased risk of breast and uterine cancer, as well as blood clotting and bleeding

-2from the uterus. Thus, there remains a need for methods of treating physical conditions that are caused by estrogen decline or lack of estrogen. There also remains a need for the treatment of such physical conditions, while reducing the side effects of hormone replacement therapy.

SUMMARY OF THE INVENTION

As a first aspect, the present invention provides the use of estrogen for the manufacture of a medicament, wherein the amount of estrogen is equivalent to generating a serum estradiol level in a subject upon transition, between 5 µg / ml and 15 µg / ml, for treating physical conditions caused by estrogen decline in that subject.

As a second aspect, the present invention provides a kit for use in consumers afflicted with or susceptible to physical conditions caused by a transient drop in estrogen, comprising:

(a) a transdermal patch for the transdermal administration of estrogen in amounts equivalent to less than 15 µg estradiol per day; and

(b) instructions describing how to use the transdermal patch to reduce the risk of osteoporotic bone fracture in that consumer.

As a third aspect, the present invention provides a further use of estrogen, for the manufacture of a medicament for transdermal administration, wherein the amount of estrogen is equivalent to less than 20 µg estradiol per day, for treating a physical condition in a subject after transition in substantial absence of exogenous progestin.

Unless otherwise defined, all technical and scientific terms used herein have their conventional meaning. The following terms have the meanings assigned to them when used in this document.

The term physical conditions caused by a transient decrease in estrogen refers to physical conditions that are common among women after menopause and which are caused at least in part by a decrease in estrogen in the body. These conditions include, but are not limited to, osteoporosis, headache, nausea, depression, hot flashes, decreased bone mineral density, and an increased risk or incidence of bone fractures, including vertebral fractures and / or hip fractures.

Transition subject refers to women in post-transition life.

Subjects affected with postmenopausal symptoms include postmenopausal women who exhibit any of the previous postmenopausal physical conditions, and in particular postmenopausal women who exhibit a decrease in bone mineral density in vertebrae, loin, or other sites, or who have been affected by fractures of either vertebrae or loin. Subjects susceptible to physical conditions due to postmenopausal estrogen decline include women achieving a severe onset of transition who show a decrease in serum estradiol levels compared to pre-transition women and post-transition women who show a decrease in serum estradiol levels but who have not yet exhibited physical conditions due to post-transition estrogen . Subjects exhibiting a decrease in serum estradiol levels include subjects exhibiting serum estradiol levels of 20 pg / ml or less, including subjects exhibiting undetectable serum estradiol levels. For the purposes of this invention, sex hormone levels, including serum estradiol levels, were measured using radioimmunoassay after extraction and column separation. The lower limit of detectability is 5 pg / ml estradiol.

Osteoporotic bone fractures refer to bone fractures, typically vertebrates or loins, for which osteoporosis is a contributing factor.

The use of the present invention is useful in the manufacture of a medicament for treating postmenopausal subjects, particularly those afflicted with or susceptible to transient physical conditions of the type discussed hereinabove. The use of the invention comprises administering an estrogen in an amount effective to produce the desired serum estradiol level in the subject. As used herein, the phrase treating physical conditions includes eliminating or reducing the extent or impact of these physical conditions in a subject afflicted with such conditions, as well as preventing the occurrence of transient physical conditions in a subject susceptible to such conditions, such as conditions caused by a transient decrease in estrogen. Although the treatment of these transient physical conditions may involve complete elimination of such conditions in subjects so affected, complete elimination of the condition is not required to fulfill the definition of a term that is within the meaning of the present invention. Thus, the present invention encompasses the use of ultra-low doses of estrogen for the manufacture of a medicament for the treatment of physical conditions caused by a decrease in estrogen and to reduce the risk of osteoporotic bone fractures in a subject

- affected or susceptible to post-passage osteoporosis.

The uses of the invention may also include the additional step of testing the serum estradiol level of the subject after the transition to be treated and determining which serum estradiol level of the subject to be treated is normal for women after the transition in a group of the same age as the subject.

In other words, the use according to the invention is not reserved for the manufacture of a medicament for the treatment of women having lower than normal serum estradiol levels than the average women after switching in the same age group. The uses of the invention can be used to manufacture a medicament for treating post-transition subjects whose serum estradiol is normal for women after the same age group. As used herein, the term normal serum estradiol level is a level of serum estradiol that is relatively close to the average serum estradiol level for women of the same age group.

The present inventors have unexpectedly found that loss of bone mineral density is not only a contributing factor leading to an increased risk of osteoporotic bone fractures in subjects afflicted with post-transition osteoporosis. Low serum estradiol levels, ie below 5 pg / ml, especially when accompanied by sex hormone binding globulin (SHBG) levels of 1 pg / dl or more substantially increase the risk of hip and vertebral fractures. In addition, a low serum level of 1,25 (OH) _{2 of} vitamin D also leads to an increased risk of hip fractures. This method of estimating the risk of osteoporotic bone fractures involves the use of logistic models developed by SAS Institute, Line, North Carolina to analyze the relationship between predictors and vertebral fractures, and proportional risk models that take into account the "case-statistical sampling" pattern to analyze the relationship between predictors and hip fractures. The analyzes are adjusted to baseline age and weight and reported as relative risks with 95% confidence intervals. Fracture ranges associated with different hormone levels are estimated using the technique described in WS Browner, American Journal of Epidemiology 123: 143 (1986), the findings of which are incorporated herein by reference in their entirety. To reduce the risk of osteoporotic bone fractures, it is believed that the risk, as measured by the use of prior art techniques, is lower for the subject being treated by the methods of the art.

Of this invention compared to the risk to the same subject prior to treatment using the methods of the invention. Thus, the risk of osteoporotic bone fractures is reduced for a subject treated with the methods of the invention as compared to an untreated subject after passing affected or susceptible to osteoporotic bone fractures.

The present inventors have also unexpectedly found that the treatment of physical conditions caused by a decrease in estrogen can be affected by ultra-low doses of estrogen without the need for progestin administration. The present inventors have now found that estrogen administration excluding progestin administration is effective in treating postmenopausal women having an uterus and ovaries. Previous hormone replacement therapies have relied on co-administration of progestin for effective treatment, especially in women who have not undergone hysterectomy or ovariectomy.

The source of exogenous estrogen in the methods of the invention may include any suitable form of administering estrogen to the subject. Suitable forms of exogenous estrogen include both natural and synthetic substances exhibiting estrogenic activity. Several forms of exogenous estrogen are commercially available. For example, suitable forms of exogenous estrogen include, but are not limited to, estradioles, including α-estradiol, 17β-estradiol, ethinylestradiol, estradiol benzoate, and estradiol 17β-cypionate; estrone; estriol; conjugated equine estrogens; and salts of the foregoing. The foregoing are all examples of steroids that exhibit estrogenic activity. Examples of non-steroidal agents exhibiting estrogenic activity include, but are not, non-steroidal agents

- 6 is not limited to diethylstilbestrol diphosphate, diethylstilbestrol dipropionate, and hexestrol. Currently, a preferred form of exogenous estrogen for use in the methods of the invention is estradiol.

The amount of exogenous estrogen to be administered to the subject is sufficient to achieve a serum estradiol level of at least about 5 pg / ml, but no more than about 20 pg / ml, and preferably no more than 15 pg / ml. In other words, according to the methods of the invention, sufficient exogenous estrogen is administered to achieve a total serum estradiol level of at least about 5 pg / ml and about 20 pg / ml. Since serum estradiol levels of an untreated subject will vary for each individual, different individuals may require administration of different doses of estrogen to achieve the desired serum estradiol level. Serum oestradiol levels of each subject being treated are not required to be increased between about 5 and about 20 pg / ml; rather, the total serum estradiol levels of each subject to be treated must be at least about 5 and no more than about 20 pg / ml. Often, the amount of exogenous estrogen to be administered is sufficient to achieve a serum estradiol level of between about 5 pg / ml and about 10 pg / ml. Contrary to the foregoing understanding, the present inventors have now found that serum estradiol levels between 5 pg / ml and 15 pg / ml advantageously cause a decrease in the risk of vertebral and hip fractures. Administration of these lower than conventional amounts of exogenous estrogen further has the advantage of decreasing the risk of undesirable side effects associated with hormone replacement therapy.

The administration of exogenous estrogen may be accomplished by any suitable route. For example, formulations for oral and parenteral administration of exogenous estrogen are known in the art and can be used in the methods of the invention. Formulations suitable for oral administration may be presented in discrete units, such as capsules, troches, or tablets, containing predetermined amounts of the active ingredient; as a powder or as granules; or as a solution, dispersion, or suspension in an aqueous or non-aqueous liquid. Such formulations may be made by any of the methods suitable in pharmacy, comprising the step of contacting the active ingredient and a suitable carrier (which may contain one or more accessory ingredients). Generally

The formulations of the invention prepare by uniformly and intimately mixing the active ingredient with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or compressing a powder or granules containing the active ingredient, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the substances in bulk form, such as powder or granules, optionally mixed with a binder, a lubricant, an inert diluent, and / or a surfactant / dispersant (s). Compressed tablets may be made by compression in a suitable tabletting machine, a powdered substance moistened with an inert liquid binder.

The amount of exogenous estrogen in the oral formulation is an ultra-low dose of estrogen which depends on the exact form of estrogen to be administered, but is typically less than 0.5 mg per day. Preferably, the amount of estrogen administered orally is between about 0.1 mg and about 0.25 mg of estrogen per day. For example, the amount of estradiol administered orally is from about 0.1 mg to about 0.25 mg per day. One skilled in the art can determine equivalent doses of other forms of estrogen.

In preferred embodiments of the invention, the estrogen is administered parenterally or transdermally rather than orally. Previous routes of administration are advantageous over oral administration, since oral administration of estrogen may lead to elevated levels of sex hormone binding globulin. Sex hormone binding globulin may exacerbate the beneficial effects of estrogen administration to subjects undergoing transition, particularly to subjects exhibiting signs of osteoporosis or loss of bone mineral density. Although oral administration is not a preferred method, the methods of the invention can be performed using oral formulations.

Formulations of the invention suitable for parenteral administration usually comprise sterile aqueous preparations of the active ingredient, these preparations being preferably isotonic with the blood of the intended recipient. These preparations can be administered by subcutaneous, intravenous, intramuscular, intradermal injection or vaginal ring. Such formulations may conveniently be prepared by mixing the active ingredient, estrogen, with water or glycine buffer,

This provides the resulting solution is sterile and isotonic with blood.

The amount of exogenous estrogen in the parenteral formulation is an ultra-low dose of estrogen which depends on the exact form of estrogen to be administered, but is typically no more than 20 µg per day. Preferably, the amount of estrogen is administered parenterally between about 5 µg and about 15 µ9 estrogen per day, and more preferably about 10 µg estrogen per day. For example, the amount of estradiol administered parenterally is from about 5 µ9 to about 15 µg per day. One skilled in the art can determine equivalent doses of other forms of estrogen.

More preferred methods of the invention include transdermal administration of exogenous estrogen. Suitable formulations for transdermal administration of estrogen are known in the art and can be used in the methods of the invention. For example, suitable transdermal patch preparations for administering exogenous estrogen are described in U.S. Pat. U.S. Patent No. 5,768,516; No. 4,460,372 to Campbell et al. U.S. Patent No. 5,768,516; No. 4,573,996 to Kwiateka et al. U.S. Patent No. 5,768,516; No. 4,624,665 to Nuwayser, U.S. Pat. U.S. Patent No. 5,768,516; No. 4,722,941 to Eckert et al., And U.S. Pat. U.S. Patent No. 5,768,516; No. 5,223,261 to Nelson et al., The disclosures of which are incorporated herein by reference in their discussion of transdermal patch technology.

A suitable type of transdermal patch for use in the methods of the invention is shown in Figure 1. A generally suitable transdermal patch 10 includes a backing layer 12 that is an impermeable, permeable surface layer 13, an adhesive layer (not shown) substantially covering the permeable surface layer. 13, and the cartridge 16 disposed or interposed between the support layer 12 and the permeable surface layer 13 such that the support layer 12 extends around the walls of the cartridge 16 and is joined to the permeable surface layer 13 at the edges of the permeable surface layer 13. The transdermal patch 10 is adhered to the skin by an adhesive layer on the permeable surface layer 13 so that the permeable surface layer 13 is in substantially continuous contact with the skin when the transdermal patch 10 is adhered to the skin. When the transdermal patch 10 is adhered to the skin of the subject, estrogen

Containing the reservoir 16 of the transdermal patch 10 is transferred through the permeable surface layer 13 from the reservoir 16 through the adhesive layer, and into and through the subject's skin. The transdermal patch 10 may optionally also include one or more penetration-enhancing agents in the reservoir 16, which enhances penetration of estrogen through the skin.

Examples of suitable materials that the support layer may contain are well known in the art of administering transdermal patches, and conventional support layer materials may be used in the transdermal patch of the present invention. Specific examples of suitable backing layer materials include, but are not limited to, a polyester film, for example, high density polyethylene; low density polyethylene or polyethylene composites; polypropylene; polyvinyl chloride, polyvinylidene chloride; ethylene-vinyl acetate copolymers; and so on.

Examples of suitable permeable surface layer materials are also well known in the art of administering transdermal patches, and any conventional materials that are permeable to the active ingredient to be administered can be used in the transdermal patch of the invention. estrogen. Specific examples of suitable permeable coating materials include, but are not limited to, a dense or microporous polymer film such as a film comprising polycarbonates, polyvinyl chlorides, polyamides, modacrylic copolymers, polysulfones, halogenated polymers, polychloroethers, acetal polymers, and acrylic resins. Specific examples of these types of conventional permeable membrane materials are described in U.S. Pat. U.S. Patent No. 5,768,516; 3,797,494 by Zaffaroni.

Examples of suitable adhesives that can be applied to the backing layer to provide an adhesive layer are also well known in the art and include, for example, pressure sensitive adhesives such as adhesives including acrylic and / or methacrylic polymers. Specific examples of suitable adhesives include polymers of acrylic or methacrylic acid esters (e.g., n-butanol, n-pentanol, isopentanol, 2-methylbutanol, 1-methylbutanol, 1-methylpentanol, 3-methylpentanol, 3-methylpentanol, 3-ethylbutanol, isooctanol, n- decanol, or their n-dodecanol esters) alone or copolymerized with ethylenically unsaturated monomers, such as acrylic acid, methacrylic acid, acrylamide,

-10-methacrylamide, N-alkoxymethyl acrylamides, N-alkoxymethylmethacrylamides, N-tert-butyl acrylamide, itaconic acid, vinyl acetate, N-branched C ₁₀ . ₂₄ alkyl maleamic acids, glycol diacrylate, or mixtures of the foregoing; natural or synthetic rubbers such as silicone rubber, styrene-butadiene rubber, butyl ether rubber, neoprene rubber, nitrile rubber, polyisobutylene, polybutadiene and polyisoprene; polyurethane elastomers; vinyl polymers such as polyvinyl alcohol, polyvinyl ethers, polyvinylpyrrolidone, and polyvinyl acetate; urea-formaldehyde resins; phenol-formaldehyde resins; resorcinol formaldehyde resins; cellulose derivatives such as ethylcellulose, methylcellulose, nitrocellulose, cellulose acetate butyrate and carboxymethylcellulose; and natural gums such as guar gum, acacia, pectin, starch, destria, gelatin, casein, etc. As will be appreciated by those skilled in the art, the adhesive layer should be inert to the active ingredient, estrogen, and should not interfere with the transdermal administration of estrogen through the permeable surface layer. Pressure sensitive adhesives are preferred for the adhesive layer of the transdermal patch to facilitate application of the patch to the skin of the subject.

Suitable penetration-enhancing agents are well known in the art. Examples of conventional penetration-enhancing agents include alkanols such as ethanol, hexanol, cyclohexanol and the like; hydrocarbons such as hexane, cyclohexane, isopropylbenzene; aldehydes and ketones such as cyclohexane, acetamide; N, N -di (lower alkyl) acetamides such as N, N -diethylacetamide, N, N-dimethylacetamide; A / - (2-hydroxyethyl) acetamide; esters such as N, N-di-lower alkyl sulfoxides; essential oils such as propylene glycol, glycerin, glycerol monolaurane, isopropyl myristan and ethyl oleate; salicylates; and mixtures of any of the above.

Figure 2 is an example of a second type of transdermal patch suitable for transdermal administration of estrogen according to the invention. In this example, the active ingredient is incorporated into the adhesive layer rather than being contained in the reservoir. Examples of these types of patches are conventional and include, for example, CLIMERA® patches available from Berlex. The transdermal patch 20 includes a backing layer 22 and an adhesive / drug layer 24. The adhesive / drug layer 24 has a

The combined function of adhesion of the patch 20 to the skin of the subject and as the carrier of the active ingredient, the estrogen to be administered. When the patch is adhered to the skin, the active ingredients are leached from the adhesive / drug layer 24 into and through the skin of the subject.

Any of the support layers described hereinabove may also be used in these embodiments. In addition, any suitable adhesive may be used as described above. The adhesive / drug layer comprises relatively homogeneous mixtures of the selected adhesive and active ingredient. Typically, the adhesive / drug layer comprises a coating substantially covering one surface of the support layer. The adhesive / drug layer may also include a penetration enhancing agent, such as those described above, by incorporating the penetration enhancing agent into a substantially homogeneous mixture of the adhesive and the active ingredient.

As will be appreciated by those skilled in the art, the transdermal patches of the invention may include various additional vehicles that are conventionally used to facilitate the transdermal delivery of the active agent, particularly the steroid active agent. Examples of such vehicles include, but are not limited to, carriers, gelling agents, adjuvants, dispersants, preservatives, stabilizers, wetting agents, emulsifying agents, and the like. Specific examples of these types of vehicles are well known in the art and any conventional transdermal patch vehicle of the invention may be used. However, it is important to note that the transdermal patches of the invention secrete progestins. Accordingly, the progestin is not a suitable vehicle for use in the transdermal patch preparations of the invention.

The amount of exogenous estrogen in the transdermal patch preparations is an ultra-low dose of estrogen which depends on the exact form of estrogen to be administered, but less than 20 gg, and typically no more than 15 gg per day is sufficient. Preferably, the amount of estrogen administered by means of a transdermal patch is between about 5 gg and about 15 gg of estrogen per day. More preferably, the amount of estrogen administered is about 10 gg per day. Although a typical estrogen dose of the method of the invention is less than 20 gg, a dose of up to 25 gg may be used. For example, the amount of estradiol administered parenterally is

12 from about 5 gg to about 15 pg per day. One skilled in the art can determine equivalent doses of other forms of estrogen. It has unexpectedly been found that the ultra-low level of estrogen used in the methods of the invention substantially reduces the risks of osteoporotic bone fractures in postmenopausal women.

Typically, transdermal patches are adapted to be worn for several days before replacement is required. Thus, the amount of estrogen in the reservoir must be sufficient to allow administration of less than 20 µg per day over a period of several days. By way of example, the transdermal patch of the invention, which is adapted to administer 10 µg of estrogen per day for seven (7) days could contain about 1 mg of estrogen. A patch suitable for administration of 15 μg per day for seven (7) days could contain approximately 1.4 mg estrogen. Based on these specific examples, one skilled in the art would be able to recognize the necessary amounts of estrogen to be included in the transdermal patch to achieve delivery of the correct daily dose of estrogen.

Preferably, the invention also provides a kit for use in a consumer afflicted with or susceptible to transient symptoms, which includes transdermal patches and instructions describing the use of these transdermal patches for treating the transient symptoms and / or reducing the risk of osteoporotic fractures of the consumer's bone. The instructions should guide the consumer to apply the transdermal patch, using its adhesive component, directly to the skin surface, such as the shoulder, to achieve transdermal administration of ultra-low doses of estrogen from the patch, thereby increasing the serum estradiol level of the consumer to between 5 pg / ml and about 20 pg / ml. The instructions could also lead the consumer to replace the patch when it is required to continue estrogen administration, if necessary to maintain this serum estradiol level using a transdermal patch. In particular, instructions to guide the consumer may replace the transdermal patch every seven (7) days to ensure administration of less than 20 µg, and preferably 10 µg of estrogen per day when a seven-day patch is used in the kit. Such kits could preferably be packaged a

-13 sold as single patches or multiple patches.

The following examples are provided to illustrate the invention and have not been construed to limit it.

These and other aspects of the invention are described below with reference to the drawings, the description of the preferred embodiment, and the examples which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a cross-sectional view of one type of transdermal patch that can be used in the methods of the invention.

Figure 2 is a cross-sectional view of a second type of transdermal patch that can be used in the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The following example and data demonstrate the correlation between serum estradiol levels and the risk of osteoporotic bone fractures, and also demonstrate the effectiveness of using ultra-low doses of exogenous estrogen to reduce the risk of osteoporotic bone fractures and to treat post-transition symptoms.

1. Experimental group

The study of osteoporotic fractures is a survey study of a statistical group of 9704 women who have been selected from population lists in four communities in the United States: Baltimore, Minneapolis, Pittsburgh and Portland. See Cummings et al., N. Engl., J. Med 332: 767-773 (1995). Women aged 65 or older were invited by post to study. The trial group excludes black women because of their low risk of hip fracture and women who have had double-sided hip replacement or who needed the help of another person while walking. Participants were asked to use estrogen, calcium supplements, and vitamin D-containing multivitamins continuously or simultaneously.

2. Clinical measurements

Mineral bone density (i.e., bone mass of calcaneum) was measured using a single photon absorptiometry obtained using an OsteoAnalyzer, Siemens-Osteon, Wahiawa, Hawaii. The mean coefficient of variation for these measurements between clinical centers was 1.2%. See, Steiger, et al., J. Bone Miner. Res. 7: 625 (1992).

3. Serum samples

All participants were instructed to follow a fat-free diet during the night and morning before the trials to minimize lipemia, which could interfere with the trials. Blood was collected between 8:00 am and 2:00 pm and the serum was immediately frozen at -20 ° C. All samples were sent for two weeks to the Biomedical Research Institute in Rockville, Maryland, where they were stored in liquid nitrogen at -190 ° C.

4. Detection of fractures

Women were contacted every 4 months by post or on periodic visits to the clinic: investigation of vital status and hip fracture was more than 99.7% complete. Hip fractures were confirmed by examination of preoperative radiograms. On average 3.3 years, 7299 women (79% of survivors) returned for repeated side spine scanning after baseline radiograms; 7238 radiogram pairs were considered suitable for assessing vertebral fractures. Radiograms were evaluated for fractures using quantitative morphometry as described in Black et al., J. Bone Miner. Res. 10: 890 (1995). A woman was classified as having a vertebral fracture if any vertebral height decreased by> 20% and> 4 mm between the baseline and the radiogram examined. See, National Osteoporosis Foundation Working Group on Vertebral Fractures, J. Bone Miner. Res. 10: 518 (1995). Those who evaluated the radiograms had no knowledge of the participants' serum test results.

5. Sampling of cases and statistical group

Using the case-statistical approach described in Prentice, RL: Biometrics 73: 1 (1986) and excluding women using hormone replacement therapy in baseline, we randomly selected serum samples from 133 participants who subsequently suffered a first hip fracture and 138 participants who had vertebral fractures. We also selected a random sample of 359 women from the statistical group, including 12 women who had subsequent hip fractures and 14 who had subsequent vertebral fractures. For hip fracture analysis, we excluded a control sample (n = 4) that suffered hip fractures prior to baseline visit; For vertebral fracture analyzes, we excluded a control sample (n = 8) that had no subsequent radiography examination or whose baseline measurement or subsequent radiography examination was technically unsuitable for morphometry.

6. Biochemical analyzes

We determined the stability of selected types of hormonal measurements in 51 women after transition by testing baseline levels and again after 3.5 years of storage at -190 ° C. The correlations (all significant at P <0.001) between the two measurements were as follows: middle region parathyroid hormone (r = 0.99), 25 (OH) vitamin D (r = 0.88), testosterone (r = 0.99), and estrone (r = 0.98). There were few, if any, deviations from the mean for these substances. The parathyroid hormone immunoradiometric assay (IRMA) was not available when baseline measurements were taken; the correlation between the levels measured by IRMA and the middle area technique was 0.78.

All trials were performed in mixed dose cases and control groups, blinded to the fracture history of participants. Samples were sent directly from the warehouse to the analytical laboratory without thawing. Initial plans required an analysis of estrone levels, but not of estradiol. After completing the first dose of tests, we added estradiol to the measurement panel and made these measurements in 89 hip fractures, 96 vertebral fractures, and 204 women who had no fracture.

Sex hormones and sex hormone binding globulin (SHBG) were

-16 tested in Endocrine Sciences of Calabasas Hills, California. Estradiol and estrone were measured by radioimmunoassay after extraction and H ₂ O column separation. Inter-assay variability for estradiol ranged from 8% to 12.5% and from 6.2% to 7.0% for estrone. The lower limit of detectability was 5 pg / ml estradiol. Total testosterone was measured by radioimmunoassay after extraction and Al ₂ O ₃ column chromatography with inter-assay variability from 6.1% to 13.4%. SHBG binding capacity was directly measured in serum using a replacement assay with inter-assay variability of 4.1% to 14.4%. The concentration of free testosterone was determined by the ammonium sulfate precipitation procedure as described in Furuyama et al., Stemids 16: 415 (1970); and Mayes, J. Clin. Endocrinol., Metab. 28: 1169 (1968). Inter-assay variability ranged from 10.7% to 15.5%.

Calciotropic hormones were tested at the UCSF Calciotropic Hormone Reference Laboratory in San Francisco, California. Intact parathyroid hormone was measured by radioimmunoassay with intra-assay variability of 5.2% and inter-assay variability of 8.5%. We measured 25 (OH) vitamin D and 1.25 (OH) ₂ vitamin D using a radioimmunoassay technique, with intra-assay variability of 6.6% for both and inter-assay variability of 15% and 24.4%, respectively. Creatinine levels were measured using an automated chemical analyzer.

7. Data analysis

We analyzed hormone and vitamin levels as continuous variables, using approximate quintiles, and when relevant as dichotomous variables (above or below the detection threshold). We used logistic models developed by SAS Institute of Cary, North Carolina to analyze the relationship between predictors and vertebral fractures and proportional risk models that took into account the case-statistical grouping (Epicure, Hirosoft International, Seattle, WA) for analysis the relationship between predictors and hip fractures. All analyzes were adjusted for baseline age and weight, and are presented as relative risks (approximated as probability ratios or risk ratios) with 95% confidence intervals. We estimated the proportion of fractures attributed to different hormone levels with standard techniques

- 17 written in Browner, Am. J. Epidemiol., 123: 143 (1986). Age-adjusted bone weights in different groups were compared using general linear models.

8. Results

Table 1 shows data collected for the characteristics of participants who suffered from hip or vertebral fractures.

Table 1

Characteristics of participants with occurrence of hip fractures, vertebral fractures and control group without consequences.

Vertebral fractures Hip fractures Fracture cases Statistical Group * Fracture cases Statistical group characteristic (N = 138) (N = 264) (N = 133) (N = 343) Average age (year) 73,2+ 71.6 75,3+ 72.1 Average weight (kg) 63,5+ 68.6 61,9+ 68.2 Contemporary smoker 12% 8,8% 17% $ 9.1% Simultaneous use of calcium supplements 53% $ 42% 51% 42% Concomitant use of vitamin D supplements 43% 42% 43% 48% Average dietary calcium intake (mg / day) 695 693 659 689 Average calcananear bone weight (g / cm ² ) 0,35+ 0.41 0,34+ 0.41

‘The number of control group for lumbar fracture analysis varies from 240 to 343 due to lack of estradiol test data. The number of vertebral fracture analysis control varies from 193 to 264; it is smaller because those who did not have the corresponding technical pairs of the basic and subsequent X-ray examination were excluded. Fracture cases selected from the statistical group are included with cases.

+ P <0.01 compared to control group $ P <0.05 compared to control group

-18 These data show that participants who suffered from hip fractures or vertebral fractures were older, weighed less, were more likely to be smokers, and had lower bone mass than the control groups.

About one third (81 out of 247) women in the randomized sample of the statistical group had undetectable estradiol levels (<5 µg / ml). These relative risk-adjusted data for weight and age are shown in Table 2 below.

Table 2

Age and weight-adjusted associations between hormone and vitamin levels and risk of hip and vertebral fractures

Relative risk (95% confidence interval) * variable incidence (%) t Hip fractures Vertebral fractures Total estradiol <5 pg / ml 32.8 2.48 (1.35 to 4.55) 2.46 (1.43 to 4.22) Sex hormones binding globulin > 0.9 pg / dl 76.4 2.04 (1.06 to 3.85) 2.27 (1.19 to 4.35) Sex hormones binding globulin (at 1.0 pg / dl) 1.25 (0.97 to 1.60) 1.49 (1.15 to 1.95) Estron> 1.5 ng / dl 77.4 1.30 (0.76 to 2.17) 1.72 (1.01 to 2.94) Free testosterone <0.7 pg / ml 19.4 1.63 (1.00 to 2.66) 1.40 (0,83-2,3S) 25 (OH) vitamin D <19 ng / ml $ 21.5 1.16 (0.70 to 1.92) 1.08 (0.64 to 1.80) 1.25 (OH) ₂ vitamin D <23 pg / ml § 15.4 2.05 (1.21 to 3.46) 1.60 (0.92 to 2.79) Intact PTH> 50 pg / ml 11.2 1.05 (0.52 to 2.12) 0.49 (0.20 to 1.17)

‘Relative risk estimated as probability ratios for vertebral fractures and risk ratios for hip fractures.

t Occurrence in a random sample of a statistical group.

$ to convert the values for 25-hydroxyvitamin D into nanomoles per liter, multiples of 2.496.

§ to convert values for 1,25-dihydroxyvitamin D to picomoles per liter, multiples

2.4.

These data demonstrate that women with undetectable estradiol levels had a 2.5-fold greater risk of hip and vertebral fractures than women who had measurable levels of this hormone. We estimated that 33% of hip fractures and 32% of vertebral fractures in this statistical group were associated with undetectable estradiol levels. These data appear to reflect a fall in the risk threshold associated with estradiol concentrations> 5 pg / ml. Women with a total estradiol level of 5 to 10 pg / ml had a 62% lower risk of hip fracture (risk ratio = 0.38; 0.20 to 0.72) and a 57% lower risk of vertebral fracture (probability ratio = 0.43; 95% confidence interval (0.25-0.76) compared to women below 5 pg / ml.

Higher SHBG levels were associated with an increasing risk of hip and vertebral fractures. After adjustment for age, every 1.0 pg / dl increase in SHBG was associated with a 1.44-fold (1.16-1.80) increase in the risk of hip fracture and a 1.65-fold (1.30-2.10) ) by increasing the risk of vertebral fracture. For hip fractures, this effect appeared to be partially mediated by weight.

In the randomized sample of the statistical group, 26% (63 of 244) had both undetectable estradiol level and SHBG level> 1 pg / dl; these women had a 14-fold greater risk of hip fracture (95% confidence interval; 3.0 to 62) and a 12-fold (3.3 to 41) greater risk of vertebral fracture than other women. Adjustment to weight slightly blunted these associations both for hip fractures (risk ratio = 6.9; 1.5 to 32) and for vertebral fractures (probability ratio = 7.9; 2.2 to 28). The associations between estradiol, SHBG and the incidence of vertebral fractures were similar in subgroups of women who had vertebral fractures at baseline and those who did not. The attributable risk for 26% of women with this combination was 60% for hip fractures and 64% for vertebral fractures.

This data from Table 2 in terms of vertebral fractures is extended to the data presented in Table 3 below.

-20Table 3

Vertebral Fracture Predictors: Multiple Variable Models *

Probability ratio (95% confidence interval) Variable (unit) modified multipremené also adapted for bone mass Estradiol <5 pg / ml 2.65 (1.50 to 4.68) 2.34 (1.30-4.22) SHBG (growth to 1 pg / dl) 1.62 (1.16 to 2.26) 1.51 (1.07-2.12) Estron> 1.5 ng / dl 2.04 (1.03 to 4.17) 2.33 (1.12 to 4.76) Age (5 year growth) 1.17 (0.89 to 1.54) 1.09 (0.82-1.44) Weight (10 kg growth) 0.71 (0.53-0.94) 0.86 (0.64-1.16) Calcaneal bone mass (decrease to SD) + 2.27 (1.52 to 3.33)

* All results are adjusted for other variables in the table. The SD (standard deviation) of the calcaneous bone mass is 0.098 g / cm ² .

+ Calcaneal bone mass was measured at baseline.

These data from Table 2 show that women with estrone values in the lowest quintile (<1.4 ng / dl) had a lower risk of vertebral fracture than women with higher levels.

Although women with lower estrone tended to have lower estradiol levels, the correlation between estrone and estradiol levels was mild (r = 0.57) and low estrone levels remained associated with a decrease in the risk of vertebral fractures after estradiol adjustment as shown in Table 3. .

Women whose vitamin D 1,25 (OH) _{2 levels} were in the lowest quintile (<23 pg / ml [<55,2 pm / lj) had and significantly increased the risk of hip fracture (risk ratio = 2.1; 1.2 to 3.5) as shown in Table 4 below.

-21 Table 4

Loin Fracture Predictors: Multiple Variable Models *

Risk ratio (95% confidence interval) Variable (unit) modified multipremené also adapted for bone mass Estradiol <5 pg / ml 2.37 (1.27-4.45) 1.92 (1.01 to 3.64) SHBG (growth to 1 gg / dl) 1.34 (0.92-1.95) 1.25 (0.85-1.84) 1.25 (OH) ₂ vitamin D <23 pg / ml § 2.20 (1.00 to 4.80) 2.16 (0.93-5.00) Age (5 year growth) 2.22 (1.50-3.28) 2.09 (1.41 to 3.10) Weight (10 kg growth) 0.56 (0.39-0.79) 0.67 (0.46-0.98) Calcaneal bone mass (decrease to SD) + 1.61 (1.03-2.56)

* All results are adjusted to other variables in the table. The SD (standard deviation) of the calcaneous bone mass is 0.098 g / cm ² .

§ To convert the values for 1,25-dihydroxyvitamin D to picomoles per liter times 2.4.

This association remains significant after adjustment for estradiol and SHBG levels (risk ratio = 2.2; 1.0 to 4.8). Further adjustment to serum creatinine caused no difference (risk ratio = 2.3; 1.0 to 5.2).

After excluding women taking vitamin D supplements, levels of 25 (OH) vitamin D (mean ± SD) were significantly higher for samples taken from January to June (28.4 ± 10.2 ng / ml [83.6 ± 25.5 nm / l]) than from July to December (25.5 ± 10.4 ng / ml [63.6 ±

25.9 nm / l], P <0.01). There were no significant differences when vitamin D levels in April-September were compared with those taken in October-March.

There were no statistically significant associations between low serum levels of 25 (OH) vitamin D and the risk of hip or vertebral fractures

-22 (see, Table 2), even after adjustment for month, period or clinical condition, or whether women taking vitamin D supplements were included or not.

The addition of bone to multiple hormone conversion models and the risk of fractures only slightly diminished the strength of association between estradiol, SHBG, and the risk of hip or vertebral fractures. See, Tables 3 and 4. Adjustment to bone mass had no significant effect on the strength of association between 1.25 (OH) ₂ vitamin D and hip fractures or between estrone and vertebral fractures.

The combined data indicate that the risk of hip and vertebral fractures in women over 65 increases substantially when serum estradiol concentrations are below 5 pg / ml, ie below the detection limit of the assay. About one third of the incidence of hip fractures and vertebral fractures can be attributed to these extremely low estradiol levels. Women with slightly higher estradiol levels, ranging from 5 to 10 pg / ml, had a much lower risk of these fractures.

Women with undetectable estradiol concentrations also had lower bone mass. However, the increased risk of fracture was slightly attenuated after adjustment to calcanean bone mass. This confirms that the association between very low estradiol levels and fracture risk is at least partially independent of bone mass.

SHBG levels also showed a strong and independent effect on the risks of hip and vertebral fractures. Women with a SHBG concentration> 1.0 gg / dl and undetectable estradiol levels were more than seven times more likely to have hip and vertebral fractures than women with lower SHBG levels and higher estradiol levels. SHBG eagerly binds circulating estradiol, so increased SHBG levels could be associated with a decrease in the bioavailability of estradiol. Thus, bioavailable estradiol concentrations may be even more strongly associated with fractures than total estradiol levels measured in this study.

Example 2

This example demonstrates a comparison of the effects of administering different amounts of estragon using a 7-day estrogen transdermal therapeutic system to prevent bone loss in postmenopausal women.

-ΣΟΙ. ways

Healthy, hysterectomized women, 45 to 65 years old, 1 to 5 years after transition with baseline mineral bone density (BMD) measured by dual energy X-ray absorptiometry (DEXA) at L2-L4 at 2.5 were selected.

SD average for normal women under 45 years old. Subjects were randomized into 1 of 4 doses of estradiol TTS (patches 6.5, 12.5, 15 and 25 cm ² delivering 0.025, 0.05, 0.06, and 0.1 mg / day estradiol) or placebo. One patch per week was worn on the abdomen during the 2-year trial. BMD at L2-L4, femoral neck and forearm, serum osteocalcin (OST) and urinary deoxypyridinoline crosslinking / creatinine (DPD) ratio were measured every 6 months. Results after 18 months of treatment are reported.

2. Results

176 women were logged, mean age 51.2 years. A total of 103 subjects completed 18 months of treatment. Prevention of loss of BMD at all doses at all time points. At 18 months, placebo-treated patients had a median percent change in lumbar BMD of -0.7 ± 4.1 vs. 3.6 ± 4.7, 3.2 ± 3.1, 3.0 ± 3.6, and 4.8 ± 5.5 for 0.025, 0.05, 0.06, and 0.1 mg / day groups, respectively. , (all differences p <0.05 vs. placebo). At 12 months, OST levels decreased from baseline in all active treatment groups (percent change -30.4 ± 42.9, -17.9 + 50.7, -13.0 ± 37.6, and -26.9 ± 29.8; all differences p <0.05 vs. placebo (-27.6 ± 60.4). DPD also decreased from baseline after 12 months in all active treatment groups (-5.8 ± 3.4, -6.0 ± 8.5, -5.1 ± 7.4, and -1.8 ± 10 changes, 8 nmol / mmol). Three of the four active treatments were significantly different from placebo. However, DPD also decreased for placebo-treated subjects.

3. Conclusions

Transdermal estradiol also available with currently available CLIMARA® TTS (0.05 and 0.1 mg / day) as well as 6.5 cm ² low-dose CLIMARA® TTS delivering

0.025 mg / day prevents transient bone loss. CLIMARA® TTS patches that deliver 10-15 gg per day are expected to have effects on skeletal bone thinning.

Example 3

The following example and data demonstrate the correlation between serum estradiol level and loss of bone mineral density. This example also demonstrates the effectiveness of using ultra-low doses of exogenous estrogen to reduce the loss of bone mineral density.

1. Experimental group

Subjects were participants in an osteoporotic fracture (SOF) study, the details of which were described in Example 1. This study is based on a randomly sampled statistical subset of 261 SOF participants who received blood and had technically equivalent calcanear BMD measured at baseline as well as at baseline subsequent examination in 1993-94. Among these, 241 women had technically adequate measurements of hip bone density in both the 1990 trials and the follow-up examinations in 1993-94. The final results are based on the statistical subgroups of 231 and 218 women with complete pairs of Calcaneum and Lumbar BMD scans who did not report concomitant use of estrogen replacement therapy during the baseline examination. Sample sizes for individual tests vary due to missing values. Sample sizes are also smaller for estradiol than for other trials because we added estradiol to the test set a little later when the study was already underway.

2. Measurements

In a baseline investigation from 1986 to 1988, a detailed questionnaire was submitted in which subjects asked about the simultaneous or prior use of estrogen, calcium, and vitamin D-containing multivitamins. Subjects were examined to obtain height and weight measurements.

Calcaneal bone mass was measured using a single photon absorptiometry at baseline and subsequent examination in 1993-1994,

-25for an average of 5.9 years. The bone densities of the lumbar and their sub-regions were measured using dual X-ray absorptiometry at the second visit from 1988 to 1990, in 82% of the original surviving statistical group. Lumbar bone density was measured repeatedly at follow-up in 1993-1994 for an average of 3.5 years. The mean coefficients of variation (CV) of 1.2% between the centers of both calcaneum and femoral neck were estimated using multiple measurements for the members of the group who visited each site. Spine bone density measurements were not performed on a sufficient number of subjects to ensure inclusion in this study.

3. Serum samples

At baseline, blood was collected between 8:00 am and 2:00 pm and sera were stored at -20 ° C. Within two weeks, samples were sent to the Biomedical Research Institute (Rockville, MD) and stored in liquid nitrogen at -190 ° C. In 1994, sera from selected participants were thawed and tested for circulating hormone levels and other biochemical measurements. Data on fracture status of participants was hidden for the testing laboratories.

Stability of measurement of selected hormones over time was determined for 51 women by baseline testing and retesting after 3.5 years storage at -190 ° C. The correlations between the measurement pairs were: estrone (r = 0.98), total testosterone (r = 0.99), intermediate region parathyroid hormone (r = 0.99), and 25 (OH) D (r = 0.88) . All correlations were significant at p <0.001. At the time of baseline measurements, immunoradiometric analysis for PTH was not available. The correlation between PTH (IRMA) and mean area for samples measured after 3.5 years of storage was 0.78. The average hormone levels between the two samples did not differ significantly.

4. Biochemical tests

Serum concentrations of estradiol, estrons and total testosterone were measured by radioimmunoassay. Sex hormone binding globulin binding capacity was measured using a replacement technique. Free testosterone levels were calculated as the product of total testosterone and percentages on SHBG

-26 unbound steroid, as determined from the ammonium sulfate precipitation procedure. Intact parathyroid hormone, 25 (OH) D and 1.25 (OH) ₂ D were measured by radioimmunoassay. Accuracy for all assays is given in Table 5, along with descriptive data on significance and standard deviations as well as quartile ranges for each hormone.

Biochemical test description

Upper Quartile ¹ | Sex Steroids & SHBG | > 3.0 I about ΛΙ appointing authority ΛΙ > 26.0 mcm ΛΙ | Calciotropic hormones and calcium > 41 | > 32 | > 40 I > 10.0 I What and 2,3 <3,0 7 <10 bCXI VI m 17.0 <26.0 Mo VI in WITH IN" WHAT 25 <32 32 <40 9.8 <10.0 OJ σ 1,6 <2,3 5 <7 | 0.9 <1.5 | 9.7 <17.0 and T " VI 23 <31 21 <25 26 <32 9.5 <9.8 Lower Quartile ¹ <1.6 <5 I <0.9 I 9.7 <1.1 <23 r— 04 V <26 <9.5 Average (SD) 2.3 (1.2) 6 (5) | 2.2 (1.9) I 20.2 (15.7) 1.5 (0.8) 34.3 (23.7) 26.2 (9.8) 32.9 (9.7) 9.7 (0.5) * from 230 168 230 230 230 231 in- WHAT CXI 229 231 Inter-assay variability (%) 8 to 12.5 6.2-7.0 | 10.7-15.5 I 6.1 to 13.4 4.1 to 14.4 8.5 15.0 12.4 1 Intra-test correlation 0.97 0.96 I 0.79 I 0.97 0.95 1 1 factor | Estron (ng / dl) | Estradiol (pg / ml) | Free testosterone (pg / ml) Total testosterone (Ng / dL) SHBG (gg / dl) | PTH (pg / ml) 25 (OH) D (pg / ml) σι Q. Q CM X g vi CM | Calcium (mg / dl)

z o c D

O T

5. Statistical analyzes

All biochemical variables except estradiol were analyzed by quartiles and as continuous variables. Because 36% of estradiol levels were less than the minimum detection level (5 µg / ml), estradiol values were divided into four groups by setting the undetectable level as the lowest category and dividing the remaining detectable levels into tertiary. Estradiol categories are listed as quartiles in the tables for simplicity. For continuous models, undetectable estradiol levels were assigned to zero.

Linear regression analysis was used to analyze the association between hormonal levels and the annual percentage change in BMD, adjusted for age and weight. The results for one-year absolute changes in BMD and including the clinical site as covariance were similar and not shown. The associations between hormone levels and bone thinning were weaker for the femoral hamster, and similar for trochanteric areas compared to total hip, and therefore we report results only for total bone loss. For age and weight-adjusted mean percent change in BMD and 95% confidence intervals were calculated using single hormone quartiles using the least-squares procedure, and a quartile trend test (age and weight adjustment) was used to evaluate statistical significance. The association between continuous hormone levels and the annual percentage change in BMD are reported as standardized beta coefficients, which represents the difference in the annual percentage change in BMD to an increase by standard deviation in the level of individual hormones.

Multiple-conversion models were designed to determine which biochemical variables were independent predictors of bone loss after control of other hormone levels. Because estradiol and estrone levels are slightly correlated in this statistical group (r = 0.64), these variables were analyzed only in separate models.

6. Results

The characteristics of the random sub-sample used in this study are shown in Table 2 and do not differ significantly from those of the statistical group from which

-29Sample was selected. The median age of women in this sub-sample was 71.3 years. The average annual bone loss was approximately 1.5 percent of the calcaneum and 0.5 percent of both the total hip and femoral neck.

Table 6

Participant characteristics for the calkanear bone loss statistical group

characteristic statistical group for calcanean bone thinning, biochemical sample Count 231 Age, years, average (SD) 71.3 (4.8) Weight, kg., Diameters (SD) 68.1 (11.7) Current calcium supplements, n (%) 102 (44.2) Current Vitamin D Supplements; n (%) 100 (44.1) Diet Calcium, mg / day, mean (SD) 687 (390) Physical activity, kcal / week, average (SD) 1698 (1791) Calcanear BMD, g / cm ² , diameter (SD) 0.409 (0.088) Total hip BMD, g / cm ² , diameter (SD) 0.765 (0.124) BMD femoral neck, g / cm ² , diameter (SD) 0.658 (0.108) Spine BMD, g / cm ² , diameter (SD) 0.850 (0.154) Calcaneal bone thinning,% / year, mean (SD) 1.48 (1.80) Total hip loosening,% / year, mean (SD) 0.49 (1.49) Femoral neck bone loss,% / year, mean (SD) 0.50 (1.70)

Hormone associations with calcaneous BMD changes are listed in the Table

7. In age and weight adjusted linear regression models, only SHBG was a significant predictor of calcanean bone loss.

Table 7

Hormone associations with calcaneous BMD changes Adjusted ^1- year average BMD change (95% Cl)

Upper quartile CD cT 1 UI T “1 ABOUT τι -1.0 (1.71 to 0.3) 1 -1.2 (-1.6, -0.7) 1-1.3 (-1.8, -0.9) -2.2 (-2.9, -1.6) ** | -1.6 (-2.1, -1.2) | 1-1.5 (-2.0, -1.0) 1 1-1.3 (-1.8, -0.8) -1.5 (-1.9, -1.1) 1 WHAT CM WHAT what what CM > »X CD 1 about' 1 about 1 τι X " 1 ABOUT 1 1 about' 1 CO O o 'cxí σ> cm r- ~ 2.2. 2.0. what_ -2.1. cm from w ¹ CD WHAT what_ CM b- what WHAT h * m; T " 1 1 1 1 1 1 1 Y " 1 1 about CD about' M b- about M ^- CM CM IN 1 ABOUT 1 IN T about 1 1 1 1 1 CM σ σ> CD σ> what it σ> n about' in' T " yy T ' cm 1 τι T cn 1 CN 1 CN 1 and what_ mF cd_ CM it oo cq ’X» ** CO T " 1 1 IN 1 1 ▼ 1 1 1 T ___ - - SL WHAT about b- 00 00 00 σ> T T " T " t ' about about about about about > 1 y ' about xr σ> _ to_ b- b-_ 00_ 00th CM CM CM η ^- η- '* ·· ** - I the. x. Stax x »> x- To h * - 1 -1.6 D) 1 -1.5 -1.2 WHAT IN" 1 CN IN" -1.3 5Γ 1 Q + K * z-x ω (0 * s? 0.5 0.4 0.2 CO o 0.2 about 0.2 0.3 C about cm cm WHAT cm cm Φ E N about about' about about WHAT about ABOUT about about TJ about «* N O IX CM o ' CM o ' about o o -0.6 ( 0 * 0 I about 1 o o about' E 2 CJ 0 (Q m T3 x F CD n ω "N> C >. eyes Q. C Ό >> C '2 F F steroid X- .about <n <> g / dL) (Pg / ml toaster estos 5 ABOUT) o SL · CD C E □) Q. CD Q. Q CM mg / dL) né C about (Λ (D 1 P σ> Q X alcium ( u. ABOUT (Ό ohlavy party Stradi >>. C about ELKOV HBG ALCIONE Q. X 1- X O * t and m , 25 (0 LL CL lu LU > ω ω CL CM

-31 Women with SHBG levels in the upper quartile (> 2.4 pg / dl) had twice as much annual calcananear bone loss (mean change = -2.2 percent; 95% Cl = -2.9%, -1.6% ) compared to women with lower quartile levels (<1.1 pg / dl; mean change = -1.2%; -1.6%, -0.7). This association remained strongly significant after adjustment to serum estradiol, estrone and testosterone levels (p <0.002). Trends towards increased calcaneous bone loss with decreased hormone levels are evident for all sex steroids (see, Table 7), but none were statistically significant. There is a boundary (p <0.10) of the inverse statistical association between serum estrone concentration modeled as a continuous variable and calcanean bone thinning.

These data with respect to the association of hormones with BMD with total hip changes (see, Table 8) demonstrate that lower levels of both estrone and estradiol were significantly associated with greater lumbar bone loss.

-32Table 8

Association of hormones with BMD changes of total hip

Adjusted ^1- year average BMD change (95% Cl)

Upper quartile 0.1 (-0.5.0.3): bo ‘o o 1 about' 0.3 (0.6.0.1) | | -0.1 (0.5.0.3) 0.7 (-1.2, -0.1) | -0.3 (-0.7.0.1) | | -0.1 (-0.5, 0.3) § | -0.4 (-0.7.0.0) x— * O o ‘b-‘ o what o 1 CXL ' CXL ' WHAT CM WHAT CM T " IN" about about about' 1 about 1 about 1 about' 1 about 1 ABOUT 1 about T m b · ' · b- Ό b · ' about' ABOUT) oT about ABOUT about about x- ' in- T IN about' about' in X-X s.X X-X xj- ,. WHAT WHAT b- WHAT b- WHAT and it it ABOUT 1 about ABOUT about 1 about 1 about 1 about' about' 1 o 't ___ x-x x-p m CM WHAT XX O CM CM CM M- ABOUT 1 ABOUT 1 about 1 ABOUT < about ABOUT 1 ABOUT 1 ABOUT 1 ABOUT 1 CM r- cm ' o> about b · ' about' ABOUT) o> ▼ - ABOUT in in about T about T about about t > 2> > 2x xix > 2 ^ s2 «* χ2χ h- WHAT it WHAT M WHAT WHAT WHAT b- about about T ABOUT 1 about 1 ABOUT about 1 about 1 about 1 about 1 Έ 03 F? Whose y- about - about about' about about" 1 about about 1 about ABOUT about about" >> » C ABOUT , 8 (1,2, - 8 (-1.2 - about 1 WHAT 6 (0.9, - 4 (-0.7, to oF 7 (1,1, 5 (-1,0, - .4 (-0.7, about ABOUT 1 o ^- ABOUT 1 about about 1 ABOUT* 1 about 1 about ABOUT 1 about + they » they » tín (-0.2, 0.2) part to S gain * o o o m o " o o 0.0, 0.5) CO O τΟ o 'xf o' 0.0, 0.4) 0.0, 0.4) (0.0, 0.3 N x »/ CM CXI WHAT t " CM CM ABOUT CM O Dí about ABOUT ABOUT about about about' about' about' about b- ABOUT b- b- b- it it WHAT WHAT from WHAT T IN" y- T " CXI CM CM CM CM CM CM CM E D ABOUT 0 ro m x E ^- • t! ABOUT) (D it O) o. C > 1 c from C «Π sexual steroids uo. 'ABOUT fll ILUJ / ml actuator stron (ng / dl) stradiol (pg / ml other testo-sti elkový testosti HBG (gg / dl) alciotropic him TH (pg / ml) 5 (OH) D (pg / m O) a ABOUT CM X O m CM Alcium (mg / dl) LL LU L1J > U it CL CM x £

For example, after adjustment for age and weight, women with estradiol levels of 10 pg / ml or higher had a slight increase in bone mass at follow-up (mean annual change = 0.1 percent; -0.5, -0.7). whereas women with estradiol levels below 5 pg / ml had above-average loin bone rates (average annual change = -0.8 percent; -1.2, -0.3)

Examining the average annual percentage change in hip density with testosterone level quartiles provides a threshold: women with total testosterone levels of 26 ng / dl or higher (upper quartile) had an average annual percentage change of only -0.1% (-0.5%, 0, 3%) compared to -0.6% (-0.8%, -0.4%) versus women below 26 ng / dl (quartiles 1 to 3 associated). This difference was statistically significant (p <0.05). Increased bone loss of the loins through the SHBG quartiles was observed, although this trend was not statistically significant in age and weight corrected regression models (p = 0.27).

In a multiprememic model involving SHBG, estradiol and testosterone levels in addition to age and weight, lower total testosterone concentrations and higher SHBG concentrations were independently associated with changes in BMD of the lumbar region. These data are shown in Table 9.

Table 9

Multipremed model: Sex hormones, SHBG and total hip loss

Difference in percent change in BMD * of lumbar

factor Adjust only for age and weight + Multiple-variable model ++ SHBG (Rise to SD) -0.3 (-0.6, 0.0) $ -0.3 (-0.6, 0.0) $ Estradiol (increase to SD) 0.2 (0.0, 0.5) 0.1 (-0.1.0.4) Total testosterone (> 25 ng / dl vs. others) 0.6 (0.0, 1.2) $ 0.6 (0.0, 1.3) $ * All analyzes include only women who have me and estradiol measurements p

comparability.

+ the first column presents the results for separate regression models for individual hormones adjusted for age and weight.

The -34 ++ second column represents the results of a single regression model containing SHBG, estradiol, total testosterone, age and weight.

$ p <0.05.

Because there were a significant number of women who did not have estradiol measurements, it was unclear whether the significant association between SHBG and hip thinning in the multipremenoid model is due to adjustment to other hormone levels or whether a stronger association between SHBG and bone thinning exists in this substatist group of women. Therefore, the age and weight-adjusted association between SHBG and hip thinning was re-examined among women who had estradiol measurements and were found to be statistically significant. See Table 9.

Expressed as a continuous variable, estradiol was not significantly associated with changes in lumbar BMD either before or after treatment for testosterone and SHBG. However, the test for the trend across estradiol quartiles remained significant after control for SHBG and testosterone levels, while the trend across estrone quartiles had marginal significance (p <0.06).

Only 25 (OH) D levels were significantly different during the winter period (January to June) compared to the rest of the year (July to December), with average values of 24.0 during winter and 26.9 otherwise (p = 0.01). There were no significant differences in calcium hormone levels when comparing measurements taken from May to October compared with November to April.

No levels of calcium hormone or calcium were significantly associated with a change in calcaneous BMD after age and weight control. See, Table

7. This remained true even after adjustment for the season (July to December versus January to June) and the clinic, and either after adjustment or exclusion of current users of calcium or vitamin D-containing multivitamins.

Lower levels of 25 (OH) D were significantly associated with increased lumbar bone thinning. See, Table 9. Women with 25 (OH) D levels in the upper quartile (> 32 pg / ml) had an annual average BMD change of -0.1 percent (-0.5, 0.3) compared to -0.7 (-1.1, -0.4) in women with 25 (OH) D levels in the lowest quartile (<21 pg / ml). This trend remained significant after adjustment to the clinic, season and use of calcium and vitamin D-containing multivitamins.

-35 There was no consistent association between parathyroid hormone (PTH) levels and bone thinning from either site. See, Tables 7 and 8. Trend testing through PTH quartiles revealed an unusual pattern with greater hip thinning for central range PTH levels (second and third quartiles) and less bone thinning in women with multiple extreme values (lower and upper quartiles).

7. Conclusion

These data demonstrate that lower serum estrogen levels are significantly associated with increased lumbar bone thinning in older women, even after adjustment for age, weight, and SHBG and testosterone levels. Indeed, women with estradiol levels of 10 pg / ml or more had an average small increase in bone mass at follow-up (mean annual change = 0.1%; 95% Cl = -0.5, 0.7), while women with undetectable estradiol levels (<5 µg / ml) lost bone from the lumbar at a rate of nearly one percent per year (-0.8%; -1.2, 0.3). Since bone loss may be an important risk factor for hip fractures in older women, these findings confirm that even moderate increases in circulating hormone levels can be effective in reducing bone loss rates and fractures.

SHBG concentrations were strongly associated with both calcaneous and lumbar thinning, independent of age, weight and sex hormone levels. SHBG binds serum estradiol and testosterone with great affinity, and this association may reflect an increased risk of bone loss with decreased levels of bioavailable sex hormones. However, the strength of this association, even after adjustment to serum estradiol and testosterone levels, confirms that there may be other effects of SHBG on bone.

Lower 25 (OH) D levels are associated with faster bone thinning from the hip but not from calcaneum. Previous results of the cross-sectional study were mixed: in two studies women after transition with lower 25 (OH) D levels were found to have lower bone mineral density (see, Villareal, J. Clin. Endocrin. Metab. 72 (3): 628-). 634 (1991); Martinez et al., Calcified Tissue Intemational 55 (4): 253-256 (1994)), but at least one other cross-sectional study found no significant association between 25 (OH) D and the lower bone mineral density, medium arc or

-36 in the lumbar spine (see, Tsai et al., Calcified Tissue Intemational 40 (5): 241 243 (1987)). However, the study by Tsai et al. had a wide age range (33-94) and did not list associations separately for older women.

PTH levels were not associated with thinning of calcaneum or hip bones in our study. This was true even after adjusting for the season and excluding women taking calcium or vitamin D supplements.

This study is a exploratory study, measuring the rates of bone mass change rather than cross-sectional bone mass measurements. Serum samples were obtained before measurement of bone mass change. The sample size was significantly larger than in most other similar studies, and subjects were sampled from a statistical group of older women living in four separate communities in the United States.

Finally, the results demonstrate that SHBG and endogenous estrogens are important determinants of bone loss in older women. Lower 25 (OH) D levels are associated with faster bone thinning from the hip but not from calcaneum. PTH and other calciotropic hormones do not significantly affect the bone loss of older women.

Example 4

The following example and data demonstrate the correlation between serum estradiol levels and bone density.

1. Experimental selection

Subjects in this analysis were participants in the osteoporotic fracture (SOF) study described in Example 1.

All 9704 SOF participants were examined and examined at one of the clinical centers during a visit to the baseline examination in 1986-1989. During this visit, detailed data was collected on the physician diagnosed with the medical condition and past medications applied. We have received standardized assessments of height, weight and strength. We measured the BMD of Calcaneum and in the proximal third of the arc using single photon absorption sensing. Lateral radiograms of the thoracic and lumbar spine were obtained. Serum samples were collected from each participant.

-37The participants completed questionnaires annually and had 3 follow-up examinations at a two-year interval at the clinic. In the first follow-up (performed 1988 - 1990), we measured the BMD of the proximal end of the femur using dual X-ray absorptiometry (DXA). Details of these measurement methods and quality control procedures for densitometry are described in Steiger et al., J. Bone Miner. Res. 7: 625-632 (1992).

2. Serum samples and biochemical tests

All participants were instructed to observe a fat-free diet overnight and to minimize lipemia in the morning during examination that could interfere with the trials. Blood was collected between 8am and 2pm and the serum was frozen at -20 ° C. Within 2 weeks, all samples were sent to the Biomedical Research Institute (Rockville, MD) where they were stored in liquid nitrogen (approximately -190 ° C). The long-term stability of these samples was determined by comparing estradiol results obtained after 2 weeks of storage at -20 ° C to those obtained after 3.5 years of storage at -190 ° C; for 51 samples, the correlation coefficient was 0.94, and the means (± SD) were 11.8 (9.0) after 2 weeks and 10.9 (9.0) after 3.5 years.

Biochemical assays using baseline serum from 400 randomly selected participants were available in the initial study group studied. For this analysis, we excluded women for whom we had no serum estradiol measurements (n = 134) and those who reported concomitant use of systemic estrogen therapy (n = 39); 247 women remained for this analysis.

The following biochemical analyzes were performed for each subject: Calcitropic factors including calcium, 25-hydroxyvitamin D, 1,25-dihydroxy vitamin D, and parathyroid hormone; growth factors including insulin-like growth factor-1 and insulin-like growth factor-binding protein 3; bone formation markers including bone-specific alkaline phosphatase and osteocalcin; sex and adrenal hormones including estradiol, estrone, testosterone (total and free), SHBG, dehydroepiandrosterone sulfate (DHEAS); other endocrine assays including thyroid-stimulating hormone (TSH). Methods for these biochemical analyzes are described in Reginster et al., Calcif. Tissue Intl, 51: 340-343 (1992). immunoassays

-38 for estradiol were performed at the Endocrine Sciences Laboratory (Tarzana, CA), and used highly sensitive assays with a 5-µg / ml lower detection limit.

To verify our findings in an independent statistical group, we measured a baseline serum oestradiol assay for a second random sample of 222 SOF participants who met the same inclusion and exclusion criteria as the initial sample analyzed. Serum oestradiol measurements in the second statistical group were performed at the Corning Nichols Institute (San Juan Capistrano, CA), and used highly sensitive assays with a 2-µg / ml lower sensitivity limit. Results from 22 split-serum samples measured in 2 reference laboratories showed similar results (correlation coefficient = 0.9, slope 1.3).

3. Determination of vertebral deformity

Radiographic morphometry was used in the baseline examination to diagnose normal vertebral deformity; the normal deformity criterion was a> 3 SD reduction in any front, middle or posterior vertebral height of at least 1 vertebra. See, Black et al., J. Bone Miner. Res. 10: 890-902 (1995).

4. Statistical analyzes

The analyzes were conducted at the SOF Coordinating Center at the University of California, San Francisco.

In the first sample (n = 247), women were divided into 4 groups based on their serum estradiol levels: in one group all women with undetectable estradiol (<5 µg / ml, n = 81, 32.8%) were included; the remaining women were divided into 3 roughly equal groups based on estradiol levels of 5-6 pg / ml, 7-9 pg / ml and 10-25 pg / ml; the number of subjects in these groups was 55 (22.3%), 63 (25.5%) and 48 (19.4%). In the second (verification) sample (n = 222) the same serum estradiol limits were used; the proportion of entities in these groups was 25.2%, 27.5%, 24.8% and 22.5%.

The consequences were investigated after adjustment to the subject's weight and age. Because weight and BMI are highly correlated and because weight shows a stronger relationship with BMD. See, Bauer et al., Ann. Intern. Med 118: 657-655 (1993). We chose the weight on

-39Integration into other subsequent models. We then evaluated the possible contribution of covariance by first investigating variables that we considered potential predictors of bone density or osteoporotic fractures. If any potential parameter showed a consistent trend (analysis of variations with a linear trend test) across estradiol groups, we further evaluated its effects in multiple model models, which resulted in bone densities at the four skeletal positions studied. These models included age, clinical location and serum estradiol as well as candidate predictor variable. If the candidate variable was captured in most of these models, it was considered a parameter. We also added pressure and smoking force to the model, as these variables were found in our earlier studies as predictors of BMD (see Bauer et al., Ann. Intern. Med 118: 657 - 665 (1993)) and fractures (see Cummings et al. , N. Engl, J. Med 332, 77-773 (1995)).

Using the variance analysis, the adjusted mean BMD for each estradiol group was calculated. The Dunnett's test was used to determine the statistical significance of the difference between the modified BMD of the lower group (undetectable estradiol) versus each of the 3 higher groups. The logistic regression model was used to calculate a multiple-adjusted probability ratio (OF) and a 95% confidence interval for the risk of normal vertebral deformity for each of the 3 higher estradiol groups versus the lowest group. Analyzes were performed using SAS (SAS Institute, Lines, NC).

5. Characteristics of subjects at estradiol level

Clinical measurements of women in this study are shown in Table 10.

-40Table 10

Clinical and laboratory values measured in 247 elderly women grouped by serum estradiol levels

Serum ESTRADIOL LEVEL undetectable 5-6 pg / ml 7 to 9 pg / ml 10-25 pg / ml P value variable (n = 80) (n = 55) (n = 63) (n = 48) Linear trend Average (SD) Age, years 71.8 (4.8) 72.9 (4.8) 71.1 (4.8) 71.8 (5.7) 0.57 Height, cm 158.9 (6.1) 159.9 (6.1) 158.1 (5.4) 159.1 (6.3) 0.84 Weight, kg 65.2 (10.3) 65.0 (11.3) 69.9 (11.3) 77.3 (15.4) <0.001 Shank / hip circumference ratio 0.81 (0.08) 0.82 (0.06) 0.81 (0.06) 0.83 (0.08) 0.08 Compression force, kg 21.6 (4.5) 20.5 (4.6) 22.8 (4.7) 21.5 (5.3) 0.48 Estron level, pg / ml 16.8 (8.7) 18.3 (8.6) 25.3 (7.4) 38.4 (13.6) <0.001 Testosterone level ng / dl 13.9 (11.1) 16.7 (9.6) 20.5 (15.5) 27.0 (15.3) <0.001 Osteocalcin level ng / ml 25.8 (10.5) 25.2 (9.1) 24.9 (9.8) 23.0 (12.7) 0.7 SHBG level, μς ^ Ι 1.7 (1.0) 1.6 (0.9) 1.4 (0.7) 1.4 (0.6) 0.01

Continuation of Table 10

undetectable 5-6 pg / ml 7 to 9 pg / ml 10-25 pg / ml P value variable (n = 80) (n = 55) (n = 63) (n = 48) Linear Trend percentage Not good health 21 11 11 15 0.22 > 1 fall last year 33 20 26 29 0.59 Smoking, Contemporary 7 9 5 9 0.11 Calcium intake <400 mg 11 20 14 4 0.32 Diabetes mellitus 9 9 10 13 0.52 Use of thiazide, concomitant 23% 15% 27% 36% 0.07 The use of thyroid hormones, concurrent 7 17 11 15 0.28 oestrogen use> 10 years prior to study 8 11 10 11 0.69

-41 On average, women in this study were about 72 years of age (Table 10). Mean estradiol varied with age; women aged 65-74 years had a mean (SD) of estradiol of 5.8 (5.1) pg / ml compared to 6.3 (4.9) pg / ml for women> 80 years. The height did not differ over estradiol groups. Body weight was statistically significantly higher among women in the upper estradiol group. Biochemical assays that did not show large or consistent differences across estradiol groups including calciumotropic factors, growth factors, bone-specific alkaline phosphatase and DHEAS (results not shown). Estrone and total testosterone were about 2-fold higher in women with upper estradiol levels compared to women with undetectable estradiol. Multiple variables, including body weight variables, variables of significant and consistent difference across estradiol groups. Osteocalcin had a downward trend at higher estradiol levels; for the upper group it was 10.9% lower than for the lowest (undetectable) estradiol group. Weight and SHBG were included in the model along with age, compression force and concomitant smoking, as they also show differences across the estradiol group and very consistent relationships to the 4 BMD sequelae.

Age and weight-adjusted baseline BMD examinations of all four skeletal sites showed a similar upward trend with higher estradiol levels. Compared to women with <5 µg / ml serum oestradiol, women with levels of 10 to 25 µg / ml had a statistically significantly greater mean BMD at all skeletal sites; the difference was 4.6%, 6.1%, 5.8% and 7.1% for the total hip, calcaneum, proximal arch and spine (p <0.05 for each comparison). After multiple adjustments, the difference remained statistically significant: 5.7%, 6.3%, 6.5%, and 6.9% for the total hip, calcaneum, proximal arch and spine.

Women in the validation study showed similar characteristics to the women in our initial study, including serum estradiol levels. Similar associations were found between the serum estradiol level and BMD in the validation statistical group. Age and weight-adjusted BMD for all four skeletal sites showed a trend similar to the original statistical group. Compared to women who had <5 pg / ml serum estradiol, women with levels of 10 to 25 pg / ml had 4.9%, 9.6%,

7.3% and 6.8% greater BMD for total hip, calcaneum, proximal arch and spine. After multiple adjustments, the difference remained statistically significant: 3.8%, 7.0%,

5.4%, 6.9% greater BMD for total hip, calcaneum, proximal arch and spine.

Women in the lowest estradiol group (<5 µg / ml), 30% had> 1 occurrence of vertebral deformities in contrast to a lower incidence in the other three groups ranging from 7 to 19%. After adjustment for age and weight, the occurrence of vertebral deformities was 60% less likely among women with estradiol levels between 5 and 25 pg / ml compared to women who had undetectable estradiol levels (OR = 0.4, 95% confidence interval ( Cl) 0.2-0.8); this ratio was minimally affected by multiple treatment (OR = 0.4, Cl 0.2 to 0.7). There was no trend towards an association between the estradiol level and the occurrence of vertebral deformities in the validation statistical group; these were similar in all groups and ranged from 15% to 19%.

6. Conclusions

These data demonstrate that serum estradiol levels below 5 µg / ml are unfavorable to the skeletal health of older women. After adjustment for age and weight, these data indicate that women with estradiol <5 µg / ml had significantly less BMD at all skeletal sites. In addition, these data show that osteocalcin, an indicator of bone turnover, has an increasing trend in women with lower serum estradiol levels. Thus, low estradiol levels exerted clinically important effects on the skeleton of older women. A similar explanation is that estradiol, when present at a low concentration, reduces skeletal remodeling, allows better bone quality and weight, and thus reduces fracture range.

The difference in BMD we observed was substantial and corresponded to about 0.4 standard deviations (SD) even after adjustment for multiple factors. This difference would be expected to reduce by 30% the risk of hip fracture (assuming 1.0 SD = 0.11 g / cm ² and 1.0 SD difference = RR 2.8). Using similar calculations, we determined that a spine density gain associated with serum estradiol of 10 to 25 pg / ml, equivalent to about 0.4 SD, would be expected to reduce by 23% the risk of spinal fracture (assuming 1.0 SD = 0 , 17 g / cm ² and 1.0 SD difference = RR 2.1).

Estron has been extensively investigated as a predictor of skeletal health among women after transition. BMD was found to be cross-linked to serum estrone levels in both white and black women (see, Cauley et al., Am. J. Epid. 139: 1035-1046 (1994)). However, this study was limited because estradiol levels were below the detection limit in more than half of the women. Estron was not predictive of hip fractures. In women after transition, estrone is a quantitatively predominant estrogen and is mainly produced from the conversion of adrenal androstenedione. Estradiol is produced by estrons and by flavoring ovarian and adrenal testosterone, which is obtained from the conversion of androstenedione and DHEA. (See, Cauley et al., Am. J. Epid. 139: 1035-1046 (1994)). These data show a relatively high correlation between serum estrone and estradiol levels both in the initial sample (r = 0.65) and in the verification sample (r = 0.78). Estradiol, not estrone, is considered to be an effector hormone at the nuclear receptor. (See, Grodin et al., J. Clin. Endo. Metab. 36: 207-214 (1973)). Estradiol is also 4 to 10 times more potent than estrone. Thus, estradiol, which was the main sex steroid hormone, had a strong, consistent and positive relationship to the skeletal consequences that could therefore be expected.

Estradiol could cause beneficial skeletal effects through several possible mechanisms: it decreases activation of bone metabolic units; antagonizes stimulation of bone resorption by parathyroid hormones; may enhance osteoblast survival by local cytokines or other growth factors; both enhances the absorption of gastrointestinal calcium and maintains renal calcium. Some or all of these actions may be amenable to very low estradiol levels. Our data also support the estradiol effect on reducing bone turnover. Indirectly, estradiol could affect bones through body weight, but our data has been adjusted to body weight and a strong association has been found.

In the past, the study of estradiol effects has been bound by insensitive tests. Only very sensitive and precise methods can distinguish between low (<30 pg / ml) and very low (<5 pg / ml) estradiol levels. Few commercial laboratories provide estradiol assays having lower detection limits below 20 pg / ml. The lack of such sensitive tests may hamper others in finding these associations. The effect of low versus very low endogenous estradiol production is subtle and could take many years to manifest differences in BMD. Our cross-sectional data confirm that estradiol levels do not decrease with aging after 65 years.

Our results indicate the protective effect of low estradiol levels against low bone mass and fractures; this effect is consistent because it can be observed for bone densities at multiple skeletal sites, for hip and calcaneum thinning, and for the risk of spinal fractures. These findings were reproduced using different laboratory highly sensitive estradiol assays. Estradiol beneficial skeletal effects in the elderly occur at levels previously considered physiologically ineffective.

The foregoing description of preferred embodiments and examples are illustrative of the present invention and are not to be construed as limiting thereof. The invention is defined by the following claims and equivalents of the claims incorporated herein.

Claims (21)

Use of an estrogen for the manufacture of a medicament, wherein the amount of estrogen is equivalent to generating a serum estradiol level in a subject upon transition, between 5 µg / ml and 15 µg / ml, for treating physical conditions caused by a decrease in estrogen in that subject. Use according to claim 1, wherein the physical conditions are selected from the group consisting of post-transient estrogen decline, an increased risk of osteoporotic bone fractures caused by post-transient osteoporosis, and loss of bone mineral density. The use of claim 1, wherein the amount of estrogen is equivalent to producing a serum estradiol level in the subject of between 5 pg / ml and 10 pg / ml. Use according to claim 1, wherein the estrogen is estradiol. The use of claim 4, wherein the medicament is in a form for oral administration. The use according to claim 5, wherein the daily dose of estrogen is less than 0.5 mg. Use according to claim 5, wherein the daily dose of estrogen is 0.1 to 0.25 mg. The use of claim 4, wherein the medicament is in a form for parenteral administration. The use of claim 8, wherein the daily dose of estrogen is less than 20 M9- The use of claim 8, wherein the daily dose of estrogen is 5 to 15 gg. The use of claim 8, wherein the daily dose of estrogen is 10 gg. The use of claim 4, wherein the medicament is in a form for transdermal administration. The use of claim 12, wherein the daily dose of estrogen is not more than 15 µg. Use according to claim 12, wherein the daily dose of estrogen is 5 to 15 µg. 15. A kit for use by consumers afflicted or susceptible to physical conditions caused by a transient drop in estrogen, comprising: (a) a transdermal patch for the transdermal administration of estrogen in amounts equivalent to less than 15 µg estradiol per day; and (b) instructions describing how to use the transdermal patch to reduce the risk of osteoporotic bone fracture in that consumer. Use of estrogen, for the manufacture of a medicament for transdermal administration, wherein the amount of estrogen is equivalent to less than 20 µg estradiol per day, for treating a physical condition in a subject after transition in the substantial absence of exogenous progestin. The use of claim 16, wherein the physical conditions are selected from the group consisting of transient estrogen decline, increased risk of osteoporotic bone fractures caused by transient osteoporosis, and loss of bone mineral density. The use of claim 16, wherein the estrogen is administered prior to the subject's hysterectomy. The use of claim 16, wherein the estrogen is estradiol. The use of claim 19, further comprising the step of testing the serum estradiol level of the subject and determining the serum estradiol level of the subject being treated that is normal for women after switching to the same age group as the subject. The use of claim 19, wherein the daily dose of estrogen is 5 to 15 µg.